Immunoassays that detect HIV antigens and antibodies directed against HIV antigens in biological samples are useful to determine if a subject is infected with or has been exposed to HIV. Current assays detect HIV antigens and antibodies to HIV-1, including groups O and M, and antibodies to HIV-2. However, assays that detect the amount of analytes in a biological sample are subject to errors that can produce spurious results. There are numerous factors that can produce errors. These factors include variations in reagent concentration (e.g., due to reagent stability or instrument pipetting errors), instrument processing steps (e.g., reagent dispense volumes, incomplete aspiration after wash steps resulting in reagent dilution, incubation timing), instrument conditions (detector temperature), and sample matrix effects (e.g. serum vs. plasma).
One method for verifying the amount or volume of a sample that is analyzed in an assay is described in U.S. Pat. No. 7,141,362. The method detects the amount of human blood coagulation Factor XIII (hFXIII) subunits a and b in a sample to determine the volume of a sample, and can also be used to distinguish plasma and serum from other types of biological samples. The amount of hFXIII in a sample is detected in an immunoassay using antibodies to hFXIII subunits immobilized on solid supports.
Further, replicate analysis of samples such as calibrators, controls or patient specimens (serum or plasma) can produce inconsistent results leading to high coefficients of variation (percent relative standard deviation). In some cases, the variation can be as high as 50% or more and can be sufficient to produce a response classified as positive for some replicates but negative for others. This variability is unacceptable for critical assays such as HIV detection. This invention addresses errors in analysis that lead to high coefficients of variation.